This project synergizes with project BC 010995 (ERM phosphorylation in lymphocytes), in which we have demonstrated that LOK is an abundant basophilic in lymphocytes near the plasma membrane, which is the predominant mediator of ERM phosphorylation. Our studies of LOK, described in that annual report fit also into the broader objectives of this project. Notably, multiple lines of our analysis this year lead us to the conclusion that the kinases of the GCK family, to which LOK belongs, are going to be predominantly basophilic. This is a paradigm-shifting interpretation since most of the currently known basophilic kinases belong to the relatively distant AGC and CAMK families. Our working conclusion that they are basophilic strengthens our interest in other GCK kinases we have provisionally identified by our mass previous spectrometric analysis of membrane/microvillus fractions from lymphocytes. However, identification of the GCK members present in those fractions was ambiguous because of sequence identity of many peptides between the various kinases in that family. Therefore, we are developing realtime PCR assays for 8 members of the GCK family and analyzing relative expression in hematopoietic cells and regulated expression during their development. Moreover, we have also initiated analysis of several GCK family members using arrays of degenerate peptides, and of custom synthesized proteomic peptides. Given our interest in functional understanding of these kinases, we are engaged in exploratory collaboration on the MST1 knockout mouse. Moreover, we are generating an SLK-knockout mouse which we will be characterizing functionally(detailed under BC 010995).